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“NucC – from phage regulator to bacterial regulator?”

“NucC – from phage regulator to bacterial regulator?”. Bacteriophage P2 late gene transcription. attP. lysA lysB. orf-30. ogr. cox. Q P O N M L XYK RS VWJ I H G fun(Z) F I F II EE’ T U D C B A tin old. int. ori. a. CTD.

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“NucC – from phage regulator to bacterial regulator?”

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  1. “NucC – from phage regulator to bacterial regulator?”

  2. Bacteriophage P2 late gene transcription attP lysA lysB orf-30 ogr cox Q P O N M L XYK RS VWJ I H G fun(Z) FIFIIEE’ T U D C B A tin old int ori

  3. a CTD Ogr-dependent transcription activation of bacteriophage P2 late promoters ‘ b a NTD b Ogr s +1 -55 -35 -10

  4. BLAST search with P2 Ogr reveals numerous “hits”

  5. Fels-2

  6. P2 Ogr Wphi Ogr L-413C Ogr 186 B PSP3 Pag NucC Fels-2 Ogr RlsB LsrS Comparison of members of the bacteriophage P2 Ogr family of transcription factors Highly conserved, small Zn-binding transcription activators Found among P2-related phages, where they regulate late gene expression 3 known homologs regulating bacterial genes NucC: S. marcescens extracellular nuclease, bacteriocin 28b RlsB: Erwinia amylovora levansucrase LsrA: Rahnella aquatilis levansucrase

  7. Serratiamarcescens • Gram negative rod • Enterobacteriaceae • Found in various ecological niches • soil • water • plants • animals • Survives under extreme conditions • disinfectants and antiseptics • distilled water • salt water • Produces characteristic red pigment, • prodigiosin

  8. The Miracle of Bolsena1263

  9. S. marcescens as a human pathogen • “Operation Sea-Spray“ 1950 • US Army released S. marcescens as a biowarfare simulant over San Francisco Bay • Followed by a significant outbreak of pneumonia and urinary tract infections between Oct 1950 and Feb 1951, including one fatality • Opportunistic nosocomial pathogen implicated in wide range of infections • Respiratory tract • UTI • Meningitis • Wound (burn) infections • Septicemia • Infective endocarditis • Important pathogen of newborns and immunocompromised patients • Increasing incidence of antibiotic resistance

  10. S. marcescens as a plant pathogen • Etiologic agent of Cucurbit Yellow Vine Disease (CYVD) • Inhabits plant tissue called phloem • Causes chlorosis, rapid wilting and death of squash, pumpkin, watermelon and cantaloupe • Transmitted by an insect vector, the squash bug Anasa tristis

  11. Looe Key Reef Implication of Serratia marcescens in coral bleaching Patterson et al. (2002) PNAS 99:8725-8730 White pox is a lethal disease of the Caribbean elkhorn coral Acropora palmata. Scanning electron micrograph of the white pox pathogen (PDL100). The bacterium was identified as S. marcescens by 16S rRNA gene sequencing, carbon source utilization patterns, and standard microbiological testing White pox disease lesion on A. palmata at Looe Key Reef, FL, August 1998. (The square pin (1.61 cm2) was for calibration of image tracing software)

  12. Serratia marcescens in the headlines

  13. nucE nucD nucC + bss nucA Extracellularproteins of S. marcescens • Serratia are unique among the Enterobacteriaceae in production of a large number of extracellular enzymes and other proteins • lipases • chitinases • proteases • deoxynuclease • gelatinase • chloroperoxidase • surfactant: “serrawettin” • bacteriocins • Many of these are growth phase regulated NucC, encoded on a cryptic prophage, is a common regulator of S. marcescens nuclease and bacteriocin 28b Are there other NucC-regulated genes?

  14. Identification of genes in the NucC regulon Proteomic approach • Compare proteins (secreted, total) in NucC+ and NucC- cells on 2D gels • Characterize NucC-regulated proteins by mass spectrometry NucC+ NucC- pH 7 pH 4 pH 7 pH 4

  15. Identification of Proteins by Mass Spectrometry

  16. Identification of genes in the NucC regulon Genomic approach • Isolate mRNA from NucC+ and NucC- cells • Probe a spotted array of Serratia marcescens DNA fragments • Characterize clones showing differential regulation

  17. For either of these approaches, a genome sequence is required http://www.sanger.ac.uk/Projects/S_marcescens/ Strain Db11 Is having one strain of S. marcescens good enough?

  18. Goal of this simulation: Does S. marcescens strain Db11 contain the nucC operon and the genes that we know it regulates?

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